Vesicle- and Hepatocyte-Based Assays for Identification of Drug Candidates Inhibiting BSEP Function
Transporters play a crucial role in the uptake of endo- and exogenous molecules in hepatocytes and efflux into the bile. The bile salt export pump (BSEP; ABCB11) is of major importance for efflux of bile salts to the bile and BSEP inhibition frequently pr
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ction Transporters are crucial for the uptake of bile salts into the hepatocyte. At the basolateral side, sodium taurocholate co-transporting polypeptide (NTCP) accounts for the majority of the bile salt transport. Yet, sodium-independent transport by members of the organic anion transporting polypeptide (OATP) family also occurs and might even compensate for the loss of function by an NTCP gene defect [1–3]. At the canalicular side of hepatocytes, the bile salt export pump (BSEP; ABCB11) is of major importance for export of bile salts to the bile [2, 4, 5]. Inhibition of this transporter leads to intrahepatic accumulation of bile salts, a phenomenon that is Mathieu Vinken (ed.), Experimental Cholestasis Research, Methods in Molecular Biology, vol. 1981, https://doi.org/10.1007/978-1-4939-9420-5_4, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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typically associated with cholestasis [6]. This is reflected by the impact of altered BSEP function or expression. Certain mutations in the gene coding for BSEP lead to hereditary forms of cholestasis due to the absence of a functional transporter [1, 2, 7–9] while direct inhibition is an important mechanism of drug-induced cholestasis (DIC) [1, 4, 5]. In addition, in vitro inhibition of BSEP has been correlated with the risk of developing cholestasis in vivo [10]. The role of other export transporters in bile salt disposition such as multidrug resistance-associated protein 2 (MRP2), MRP3, and MRP4 is not fully understood [7], but these transporters appear unable to compensate for the loss of BSEP function. Several in vitro systems to study BSEP inhibition exist [11]. Suspended and sandwich-cultured hepatocytes (SCH) are more physiologically relevant than most other assays because of the presence of other transporters and metabolizing enzymes. However, their inherent complexity and the need for time-consuming hepatocyte isolation (and culturing in the case of SCH) limit high- throughput use. Other systems, such as BSEP-transfected cell lines, might be restricted by the orientation of the transporter: potential substrates and inhibitors first have to reach the cytosol before they are available for interaction with the intracellular domain of the transporter. Inside-out membrane vesicles prepared from transfected cells therefore offer the advantage of direct access to the cytosolic side of BSEP by substrate present in the incubation buffer. In addition, the high abundance of a single transporter allows high-throughput evaluation of compounds for interaction with this transporter [11]. Protocols for membrane vesicle purification have been described previously [12–15]. Briefly, cells are lysed and centrifuged several times to remove undesirable cell components. For different intravesicular conditions, the buffers used during the process can be modified. The final suspension is passed through a 27-gauge needle for further purification and the protein content is determined. After dilution, the membrane vesicles can be uti
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